WO2020078254A1 - 一种从基因工程水稻种子中分离纯化重组人生长激素的方法 - Google Patents

一种从基因工程水稻种子中分离纯化重组人生长激素的方法 Download PDF

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WO2020078254A1
WO2020078254A1 PCT/CN2019/110385 CN2019110385W WO2020078254A1 WO 2020078254 A1 WO2020078254 A1 WO 2020078254A1 CN 2019110385 W CN2019110385 W CN 2019110385W WO 2020078254 A1 WO2020078254 A1 WO 2020078254A1
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chromatography
buffer
growth hormone
recombinant human
human growth
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杨代常
董亮亮
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武汉禾元生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C07K14/615—Extraction from natural sources

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  • the invention belongs to the field of biotechnology, and in particular relates to a method for separating and purifying recombinant human growth hormone from genetically engineered rice seeds.
  • Human growth hormone is a single peptide chain protein hormone released by anterior pituitary eosinophils. It consists of 191 amino acid residues and is the most abundant hormone in the adenohypophysis (approximately the total 50% of the content). Its main physiological function is to promote the synthesis of various tissues of the human body, especially proteins, and to achieve increased functions by stimulating the growth of bone articular cartilage and epiphyseal cartilage.
  • GH human-derived growth hormone
  • Eli Lilly and Novo Nordisk have developed 191 amino acid growth hormones.
  • the natural structure is still different, the antibody production rate is high, and it is easy to bring impurities and cause allergic reactions.
  • Growth hormone which was launched in the 1980s due to the use of mammalian recombinant synthesis technology, has a low antibody production rate.
  • BSE virus due to the possibility of introducing BSE virus, it is currently used by a few manufacturers.
  • E. coli secreted gene expression technology was used to produce growth hormone.
  • the amino acid sequence is the same, the antibody production rate is low, and the biological activity is good. It is currently used by most leading international manufacturers. From the end of the 20th century to the beginning of the 21st century, various manufacturers have successively started to develop long-acting growth hormone.
  • E. coli Bacillus, yeast, mammalian cells and other expression systems can also be produced, but due to low expression levels and high costs, few companies use the above system for rhGH production).
  • E. coli Bacillus, yeast, mammalian cells and other expression systems can also be produced, but due to low expression levels and high costs, few companies use the above system for rhGH production.
  • the shortcomings of low expression and complicated process, and its clinical application also have certain side effects, such as local transient reactions at the injection site (pain, tingling, redness, etc.) and symptoms of fluid retention (peripheral edema, joint pain, or muscle Pain), in addition, long-term use may produce antibodies in the human body and affect the therapeutic effect.
  • the annual cost of recombinant human growth hormone long-acting / water injection / powder injection is 19.5, 4.25, and 20,000 yuan, respectively.
  • the high cost of treatment also limits the promotion of recombinant human growth hormone. It is estimated that there are 7 million people with short stature of 4 to 15 years old in China, and the estimated applicable population is about 4 million, and the annual applicable population is 400,000 to 500,000, and the current penetration rate is less than 2%, and the incremental penetration rate is not To 15%.
  • CN200480043565 discloses a method for purifying human growth hormone obtained by DNA recombination technology using hydrophobic chromatography technology.
  • the existing separation and purification technology of recombinant human growth hormone mainly focuses on the method of extracting rhGH from animal cells, Escherichia coli cells, yeast cells, etc.
  • CN103724425A discloses a new method for crude extraction of recombinant human growth from E. coli cells Hormone extraction method. Song Xuewen, etc.
  • hGH human growth hormone
  • the invention further studies the expression, extraction and purification of recombinant human growth hormone protein from transgenic rice seeds on the basis of rice endosperm cell biological gene engineering technology.
  • the purpose of protein purification is to separate the target protein from all components of the crude extract of seed protein, while still retaining the biological activity and structural integrity of the target protein. Separation and purification of protein need to select the appropriate purification scheme according to the characteristics of the protein and the nature of other substances to be separated.
  • the object of the present invention is to provide a method for separating and purifying recombinant human growth hormone (OsrhGH) from genetically engineered rice.
  • the method of the present invention specifically includes the following steps:
  • the crude extract of recombinant human growth hormone in step 1) is prepared by the following method:
  • step ii) Adjust the pH of the extraction mixture of step i) to 5.0 to 5.8, the preferred pH is 5.4 to 5.6, and precipitate for 1 to 4 hours;
  • step iii) Filter the mixture of step ii) and collect the filtrate to obtain a crude extract solution containing a high concentration of recombinant human growth hormone.
  • the method of the present invention includes the following steps:
  • the genetically engineered rice is hulled and processed into semi-polished rice, which is ground into 80-100 mesh rice flour.
  • the rice flour and the extraction buffer were mixed at a ratio of 1: 5 (weight / volume, kg / L), and extracted at 25 to 28 ° C for 1 hour.
  • the components of the extraction buffer are: 25 mM Tris-acetic acid, 25 mM NaAc, pH 7.5. Adjust the pH of the above-obtained mixture with 20% acetic acid to 5.4 to 5.6. After leaving the precipitate for at least 2 hours, add 2 to 5% of perlite for pressure filtration. After the pressure filtration is completed, adjust the pH to 5.0 to 5.8. After 0.22 ⁇ m After filtering, the filter is the crude extract of OsrhGH.
  • the sample loading volume is 20 ⁇ 40CV; elution is carried out at a linear flow rate of 170 ⁇ 240cm / h with 50mM Tris and 50mM NaAc buffer pH 6.0 ⁇ 6.5, and the eluent containing OsrhGH is collected to obtain the primary product I containing OsrhGH ;
  • Phenyl Bestarose HP chromatography is used for purification.
  • the column was equilibrated with a buffer solution of 4 to 6 column volumes of pH 7.2 to 7.4, conductivity of 73 to 76 mS / cm, 8 to 12 mM sodium phosphate, and 0.5 M ammonium sulfate at a flow rate of 50 to 180 cm / h.
  • the eluted sample of intermediate purity obtained in step 3 was adjusted to conductivity of 75-78mS / cm with 3mM ammonium sulfate, and pH was adjusted to 7.2-7.4 as the sample loading liquid for this step of chromatography; use 8-pH of 7.2-7.4
  • the sample was eluted at a flow rate of 50-180 cm / h with 12 mM sodium phosphate and 0.5 M ammonium sulfate buffer, and the eluent containing recombinant human growth hormone was collected to obtain recombinant human growth hormone with a purity of more than 95%.
  • the inventor relies on the company's two dominant platforms-the high-efficiency expression technology platform and recombinant protein purification technology platform of rice endosperm cell bioreactor, which uses rice endosperm cells to efficiently express recombinant human growth hormone, and the purified plant-derived recombinant human growth
  • the hormone has the advantages of high activity, high expression level, simple purification process and low cost.
  • the currently obtained genetically engineered rice can obtain about 80mg of recombinant human growth hormone per kilogram of rice. According to the market price of 60 yuan / mg (120 yuan / mg of liquid agent), the output value per kilogram of rice reaches 4800 (9600) yuan. It can effectively reduce the cost of growth hormone treatment and bring the gospel to the dwarf patients with poor families.
  • Figure 1 shows the results of SDS-PAGE (left) and Western Blot (right) detection of three extract buffer systems.
  • Figure 2 shows the binding of OsrhGH with four cation chromatography media.
  • Figure 3 shows the effect of acid precipitation at different pH values on the content of OsrhGH in the extract.
  • Figure 4 shows the effect of different pH values before and after acid precipitation on the activity of OsrhGH in the extract.
  • Figure 5 shows the optimization of the pH of MMC chromatography loading solution.
  • Figure 6 is the exploration of MMC chromatography elution conditions.
  • Figure 7 shows the optimization of MMC chromatography elution conditions.
  • Figure 8 shows the SDS-PAGE and Western Blot detection of the penetrant in the MMC chromatography load determination.
  • Figure 9 is the detection chart of penetrating fluid electrophoresis and Western Blot in the determination of QFF chromatographic load.
  • Figure 10 shows the results of Phenyl HP and Octyl FFSDS-PAGE.
  • Figure 11 shows the results of Phenyl HP chromatography optimized 1SDS-PAGE.
  • Figure 12 shows the results of SDS-PAGE of Phenyl HP chromatography conditions optimization 2 (left) and optimization 3 (right).
  • Figure 13 shows the results of SDS-PAGE of Phenyl HP chromatography eluent under different Q FF chromatography loading conditions.
  • Figure 14 shows three batches of verified chromatograms of MMC chromatography.
  • Figure 15 shows three batches of verified chromatograms of QFFFF chromatography.
  • Figure 16 shows the three batches of Phenyl HP verification chromatograms.
  • Figure 17 shows the results of OsrhGH SDS-PAGE detection after purification.
  • Figure 18 shows the results of the OsrhGH activity measurement after purification.
  • MMC Bestarose Diamond MMC
  • Phenyl Bestarose HP Phenyl HP filler
  • the manufacturer is Boglong (Shanghai) Biotechnology Co., Ltd .
  • Q Sepharose FF (QFF) filler the manufacturer is General Electric (GE Healthcare) company
  • XK 26/20, XK16 / 20 chromatography column purchased from General Electric (GE Healthcare) company
  • other materials or reagents without special instructions are conventional commercial products
  • the genetically engineered rice is hulled and processed into semi-refined rice, which is ground into 80-100 mesh rice flour.
  • the rice flour and the extraction buffer were mixed at a ratio of 1: 5 (weight / volume, kg / L), and extracted at 25 to 28 ° C for 1 hour.
  • the components of the extraction buffer are: 25 mM Tris-acetic acid, 25 mM NaAc, pH 7.5. Adjust the pH of the above-obtained mixture with 20% acetic acid to 5.4 to 5.6. After leaving the precipitate for at least 2 hours, add 2 to 5% of perlite for pressure filtration. After the pressure filtration is completed, adjust the pH to 5.4 to 5.6. After membrane filtration, it is OsrhGH extract.
  • OsrhGH extract was adjusted to pH 6.9 (pH 7.5 buffer extract is pH 6.9), 6.3, 5.8, 5.5, 5.25, 5.0, 4.8, each 3ml / tube at room temperature for 4h, after centrifugation
  • the samples of the supernatant were kept separately. Among them, pH 6.9, 5.25, 5.0, 4.8 were sent to measure the cell viability.
  • the results of SDS-PAGE and Western Blot are shown in Figure 3.
  • the SDS-PAGE results of acid precipitation samples were analyzed by Quantity Gray, and it was found that when the acid precipitation reached pH5.8, the recovery of the target protein band was the highest; when the pH value was lower than pH5.2, the target protein was obviously lost.
  • the acid precipitation conditions below pH5.2 have a significant effect on the cell activity of hGH and the recovery of the target protein; the purity of the target protein in the eluent is significantly reduced when the sample is loaded at pH 5.0, and the pH value of the sample should be at pH 5.
  • MMC chromatography is the first step of purification, and the recovery of the target protein is more important. Based on this, pH 5.5 and conductivity of 4.5 mS / cm are the preferred loading conditions.
  • the sample was eluted by QFFFF chromatography in Example 3, ammonium sulfate with a final concentration of 1M was added, and the pH was adjusted to 7.5 to be the sample loading solution.
  • the chromatographic loading solution was respectively loaded to a phenyl HP, Octyl FF chromatography column with a column volume of 5 mL.
  • the loading flow rate was 1.25 mL / min, and the loading volume was 50 mL.
  • Elution was performed by linear elution. SDS-PAGE results showed ( Figure 10) that there was almost no degradation band under the No. 8 sample of Phenyl HP chromatography (shown in Figure 10).
  • the target protein was mainly concentrated in the first half of the main peak of the elution peak, and the degradation bands No. 9 and No. 10 Began to be eluted in large quantities.
  • Octyl FF chromatography linearly elutes. Because the resolution is not as good as phenylHP, there is only one main peak. The degraded protein mainly exists in the first half of the main peak of sample No. 6. There is not much room for optimization. Therefore, Phenyl HP chromatography was initially determined as a purification step.
  • the Phenyl HP chromatography conditions were optimized as follows: (1) 1M ammonium sulfate loading. 0.5M ammonium sulfate (containing 10mM PB, pH7.5) washes, 0 ⁇ 0.5M ammonium sulfate (containing 10mM PB, pH7.5) linear elution; (2) 0.5M ammonium sulfate loading, 0.25M ammonium sulfate elution; (3) Sample loading with 1M ammonium sulfate, 0.5M ammonium sulfate, 0.25M ammonium sulfate, 0.125M ammonium sulfate (containing 10mM PB, pH7.5) gradient elution.
  • Phenyl HP chromatography selected 0.5M ammonium sulfate concentration for loading, and the eluent ammonium sulfate concentration was about 0.25M about.
  • the sample volumes of QFFFF and PhenylHP chromatography were designed as follows: (1) load QCFF with 10CV chromatography eluent according to 10CV, load PhenylHP chromatography; (2) load QFF Load 20CV chromatographic eluent to load Phenyl HP according to 5CV; (3) Load 20CV chromatographic eluent to QFF with 10CV to load Phenyl HP; the results show that QFF 20CV, Phenyl HP 5CV and QFF 10CV The eluted sample of Phenyl HP 10CV has higher purity, which is obviously higher than that of QFF 20CV and Phenyl HP 10CV (see Figure 13). Comprehensive cost and other factors, the final sample volume of QFF is 20CV, and the sample volume of Phenyl
  • the column was equilibrated with 4 to 6 column volumes of 8 to 12 mM sodium phosphate and 0.5 M ammonium sulfate buffer with a pH of 7.2 to 7.4 and a conductivity of 73 to 76 mS / cm at a flow rate of 50 to 180 cm / h.
  • the eluted samples of Q FF intermediate purity were adjusted to conductivity of 75-78mS / cm with 3mM ammonium sulfate, and pH was adjusted to 7.2-7.4 as the loading solution for this step of chromatography; 8-12mM phosphoric acid with pH 7.2-7.4 was used Sodium, 0.5M ammonium sulfate buffer was used to elute the sample at a flow rate of 50-180 cm / h, and the eluate containing recombinant human growth hormone was collected to obtain recombinant human growth hormone with a purity of more than 95%.

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Abstract

提供一种从基因工程水稻中分离纯化重组人生长激素(OsrhGH)的方法。包括以下步骤：1)从重组人生长激素基因工程水稻种子中提取含有重组人生长激素的蛋白粗提取物；2)将含有重组人生长激素的蛋白粗提取物经BestaroseDiamondMMC阳离子交换层析，得到初级产物I；3)将初级产物I经Q Sepharose FF阴离子交换层析，得到含有重组人生长激素的中级产物II；4)将中级产物II经PhenylBestaroseHP疏水层析，得到纯化的重组人生长激素目标物，纯度大于95％，具有高活性。

Description

一种从基因工程水稻种子中分离纯化重组人生长激素的方法 技术领域
本发明属于生物技术领域，具体涉及一种从基因工程水稻种子中分离纯化重组人生长激素的方法。
背景技术
人生长激素(human growth hormone,hGH)是由脑垂体前叶嗜酸细胞释放的一种单一肽链的蛋白质激素，由191个氨基酸残基构成，是腺垂体中含量最多的激素(约占总含量的50％)。其主要生理功能是对人体各种组织尤其是蛋白质的合成具有促进作用，通过刺激骨关节软骨和骨骺软骨生长从而达到增高的功能。
20世纪50年代开始将人源生长激素(GH)应用于临床，由于其源于尸检人腺垂体，提取困难且易混有病毒和其他垂体激素等物质，因此未进行大规模临床推广。之后，人们逐渐开始利用化学合成和基因重组方法生产生长激素，美国基因工程技术公司的基因重组法在1981年实现工业化。美国基因工程技术公司生产的重组生长激素(商品名为somatrem)含有192个氨基酸，比天然结构多了一个氨基酸，用于人体后会导致64％的患者出现生长激素抗体。为有效解决用药后的抗体产生问题，美国礼来和诺和诺德开发了191个氨基酸的生长激素，但因天然结构依然不同，抗体产生率较高，且容易带入杂质导致过敏反应发生。20世纪80年代上市的生长激素由于应用哺乳动物的重组合成技术，抗体产生率很低，但由于可能引入疯牛病病毒，目前被少数生产厂商使用。20世纪90年代大肠杆菌分泌基因表达技术进行生产生长激素，氨基酸序列相同，抗体产生率低，生物活性好，目前为大多数国际领先生产厂商使用。20世纪末到21世纪初，各厂家相继开始研发长效生长激素。
目前市场重组人生长激素均采用大肠杆菌表达(芽孢杆菌、酵母以及哺乳动物细胞等表达系统也能产生，但是由于表达水平低、成本高等因素，鲜有公司采用上述系统进行rhGH的生产)，存在表达量低、工艺复杂等缺点，同时其在临床应用也存在一定的副作用，如注射部位局部一过性反应(疼痛、发麻、红肿等)和体液潴留的症状(外周水肿、关节痛或肌痛)，另外长期使用可能在人体内产生抗体从而影响治疗效果。在经济层面上重组人生长激素长效/水针/粉针年花费分别为19.5、4.25、2万元。高昂的治疗费用也限制了重组人生长激素的推广使用。据估计，我国4～15岁矮小症患者达700万，预计存量适用人群约400万人，每年新增适用人群40～50万人，而目前存量渗透率不到2％，增量渗透率不到15％。
CN200480043565公开了一种采用疏水层析技术纯化通过DNA重组技术得到的人生长激素的方法。现有重组人生长激素的分离提纯技术主要围绕从动物细胞，大肠杆菌菌体，酵母菌体等提取rhGH的方法，例如，CN103724425A公开了一种新的从大肠杆菌菌体中粗提重组人生长激素提取方法。宋学文等将在中国仓鼠卵巢细胞(CHO)中基因克隆表达的人生长激素(hGH)培养物经Octyl-SepharoseCL-4B疏水层析，SephadexG-100凝胶过滤层析，DEAE-Cellulose DE52离子交换层析，得到了较纯的hGH。(天津医科大学学报，1995(2):18-20)。
发明内容
本发明在水稻胚乳细胞生物基因工程技术的基础上，进一步研究从转基因水稻种子中表达、提取、纯化重组人生长激素蛋白。蛋白质纯化的目的是将目标蛋白质从种子蛋白粗提取液的全部组分中分离出来，同时仍保留目的蛋白的生物学活性及结构的完整性。蛋白质的分离和纯化需根据蛋白的特性及待分离的其他物质性质来选择合适的纯化方案。
本发明的目的在于提供一种从基因工程水稻中分离纯化重组人生长激素(OsrhGH)的方法。
为实现上述目的，本发明的方法具体包括以下步骤：
1)从重组人生长激素基因工程水稻种子中提取含有重组人生长激素的蛋白粗提取物；
2)将含有重组人生长激素的蛋白粗提取物经Bestarose Diamond MMC阳离子交换层析，得到初级产物I；
3)将初级产物I经Q Sepharose FF阴离子交换层析，得到含有重组人生长激素的中级产物II；
4)将中级产物II经Phenyl Bestarose HP疏水层析，得到纯化的重组人生长激素目标物；
本发明所述的方法中，步骤1)所述重组人生长激素粗提取物是通过下述方法制备：
i)将含有重组人生长激素的基因工程水稻种子粉碎后与提取缓冲液按重量/体积比(kg/L)＝1∶5混合，在4～30℃下提取1～2小时；所述提取缓冲液包含5～25mM Tris，5～25mM NaAC，pH为5.5～7.5；
ii)将步骤i)的提取混合物的pH调节至5.0～5.8，优选的pH为5.4～5.6，并沉淀1～4小时；
iii)过滤步骤ii)的混合物，收集滤液，获得含有高浓度重组人生长激素的粗提物溶液。
进一步的，本发明方法包含以下步骤：
(1)将基因工程稻谷脱壳加工成半精米，研磨成80～100目的米粉。将米粉与提取缓冲液以1:5(重量/体积，kg/L)的比例混合，于25～28℃提取1小时。提取缓冲液的成分为：25mM Tris-乙酸，25mM NaAc，pH 7.5。将上述得到的混合物用20％乙酸调节pH至5.4～5.6，放置沉淀至少2小时后，加入2～5％的珍珠岩进行压滤，压滤完毕后复调pH至5.0～5.8，经0.22μm滤膜过滤后即为OsrhGH的粗提取物。
(2)采用Bestarose Diamond MMC层析介质进行初级分离纯化。采用4～6倍柱体积(CV)的pH为5.2～5.6的50mM Tris、50mM NaAc的缓冲液，以170～240cm/h的线性流速平衡层析柱；以步骤1的粗提物为层析上样液，其中上样液pH为5.2～5.6、电导3.5～5mS/cm，优选为4～5mS/cm。上样体积20～40CV；用pH为6.0～6.5的50mM Tris、50mM NaAc缓冲液，以170～240cm/h的线性流速进行洗脱，收集含有OsrhGH的洗脱液，获得含OsrhGH的初级产物I；
(3)采用Q Sepharose FF层析介质进行中级分离纯化。采用10～15倍柱体积的pH为7.0～7.5的25mM Tris-HCl缓冲液，以120～300cm/h的流速平衡柱子；将步骤2的含OsrhGH的洗脱液中加入约等体积的纯水，并调节pH至7.2～7.4，作为此步层析的上样液，其中样品电导为3～3.4mS/cm；用4～6倍柱体积、pH为7.2～7.4、电导5.5～6.0mS/cm的25mM Tris-HCl，35mM NaCl缓冲液以120～300cm/h的流速进行杂蛋白的洗脱；用4～6倍柱体积、pH为7.2～7.4、电导8.1～8.6mS/cm的25mM Tris-HCl，60mM NaCl缓冲液以120～300cm/h的流速进行OsrhGH的洗脱，获得含OsrhGH的中级产物II；
(4)采用Phenyl Bestarose HP层析进行精纯。用4～6倍柱体积的pH为7.2～7.4，电导为73～76mS/cm的8～12mM磷酸钠、0.5M硫酸铵的缓冲液以50～180cm/h的流速平衡柱子。将步骤3得到的中级纯度的洗脱样品采用3mM硫酸铵调节电导至75～78mS/cm，pH调节至7.2～7.4，作为此步层析的上样液；用pH为7.2～7.4的8～12mM磷酸钠、0.5M硫酸铵缓冲液以50～180cm/h的流速洗脱样品，收集含有重组人生长激素的洗脱液，获得纯度95％以上的的重组人生长激素。
本发明人依托公司两大优势平台——稻胚乳细胞生物反应器的高效表达技术平台及重组蛋白纯化技术平台，采用水稻胚乳细胞高效表达出重组人生长激素，经纯化后的植物源重组人生长激素具有高活性，高表达量，纯化工艺简单，成本低廉等优点。目前获得的基因工程水稻每公斤稻谷可获得约80mg的重组人生长激素，按照市场上粉针60元/mg(水剂120元/mg)计算，每公斤水稻产值达4800(9600)元。可有效降低生长激素治疗成本，为了家庭贫困的矮小症患者带来福音。
附图说明
图1为3种提取液缓冲体系的SDS-PAGE(左)及Western Blot(右)检测结果。
图2为4种阳离子层析介质对OsrhGH的结合情况。
图3为不同pH值酸沉对提取液中OsrhGH含量的影响。
图4为不同pH值酸沉前后对提取液中OsrhGH活性影响。
图5为MMC层析上样液pH的优化。
图6为MMC层析洗脱条件的摸索。
图7为MMC层析洗脱条件的优化。
图8为MMC层析载量测定中穿透液的SDS-PAGE及Western Blot检测。
图9为Q FF层析载量测定中穿透液电泳及Western Blot检测图。
图10为Phenyl HP及Octyl FFSDS-PAGE检测结果。
图11为Phenyl HP层析条件优化1SDS-PAGE检测结果。
图12为Phenyl HP层析条件优化2(左)及优化3(右)SDS-PAGE检测结果。
图13为不同Q FF层析上样量条件下的Phenyl HP层析洗脱液SDS-PAGE检测结果。
图14为MMC层析3批验证层析图谱。
图15为Q FF层析3批验证层析图谱。
图16为Phenyl HP 3批验证层析图谱。
图17为提纯得到的OsrhGH SDS～PAGE检测结果。
图18为提纯得到的OsrhGH活性测定结果。
具体实施方式
以下通过结合附图详细说明本发明的特点和优点。所提供的实施例仅是对本发明方法的举例说明，而不以任何方式限制本发明揭示的其余内容。
以下实施例中使用的Bestarose Diamond MMC(MMC)、Phenyl Bestarose HP(Phenyl HP)填料，生产商是博格隆(上海)生物技术有限公司；Q Sepharose FF(Q FF)填料，生产商是通用电气(GE Healthcare)公司；XK 26/20、XK16/20层析柱，购自通用电气(GE Healthcare)公司；其它材料或试剂如无特殊说明均为常规市售产品；
【实施例1】从基因工程稻米中提取OsrhGH
1.提取液缓冲体系的确定
分别用10mM的Tris-HCl缓冲液、10mM的PB缓冲液和10mM柠檬酸缓 冲液(pH均为7.5±0.1)提取5g米粉(1:5提取)，离心后过滤，取16μL样品进行SDS-PAGE和Western Blot检测，检测图谱如图1所示。同时初步采用ELISA法进行测定，结果显示柠檬酸缓冲液提取的目的蛋白量最多(见表1及图1)，但是当样品放置过夜后发现，柠檬酸缓冲液提取的蛋白损失严重(表1)，Tris缓冲液样品最稳定。因此最终决定采用Tris缓冲体系作为OsrhGH提取液的基本缓冲成分。并在此基础上不断优化并形成最终的提取液配方：25mM Tris-乙酸，25mM NaAc，pH 7.5。
表1 3种缓冲液提取后样品放置前后ELISA检测结果
Figure PCTCN2019110385-appb-000001
2.从基因工程稻米中提取OsrhGH
将基因工程稻谷脱壳加工成半精米，研磨成80～100目的米粉。将米粉与提取缓冲液以1:5(重量/体积，kg/L)的比例混合，于25～28℃提取1小时。提取缓冲液的成分为：25mM Tris-乙酸，25mMNaAc，pH 7.5。将上述得到的混合物用20％乙酸调节pH至5.4～5.6，放置沉淀至少2小时后，加入2～5％的珍珠岩进行压滤，压滤完毕后复调pH至5.4～5.6，0.22μm滤膜过滤后即为OsrhGH的提取液。
【实施例2】通过阳离子交换层析进行初级纯化
1.4种常见阳离子层析介质对OsrhGH结合能力比较
将OsrhGH提取液pH调节至5.8，分别上样至SP FF(博格隆)、UNO S(伯乐)、CM FF(GE)和MMC(博格隆)层析介质上，检测4种介质的流穿液，发现MMC的结合最为牢固(图2)，因此选择MMC作为第一步初级纯化的介质。
2.提取液的预处理
OsrhGH提取液分别调酸至pH 6.9(pH7.5缓冲液提取后的提取液即为pH6.9)、6.3、5.8、5.5、5.25、5.0、4.8，各取3ml/管室温放置4h，离心后，上清分别留样，其中pH 6.9、5.25、5.0、4.8送样测定细胞活性，SDS-PAGE及Western Blot结果如图3所示。对酸沉样品的SDS-PAGE结果进行Quantity One灰度分析，发现酸沉至pH5.8时，目的蛋白条带回收最高；当pH值低于pH5.2时，目的蛋白明显损失。对各酸沉样品进行细胞活性测定，结果发现酸沉对样品活性影响较小，目的蛋白降解后可能仍具有活性(图4)。结合PAGE上目的 条带的含量和活性测定结果来确定酸沉pH值，最终认为酸沉pH值不得低于pH5.25，因此，适合的酸沉pH值范围为5.2-6.9之间
3.MMC上样pH值的优化
前期的研究表明当pH为5.8时，MMC层析柱的载量约为10CV，而细胞活性测定结果证明pH5.0条件下酸沉对提取液蛋白活性无影响(图4)。因此尝试降低上样液pH值，以提高载量。故分别设置pH5.0，电导4.5mS/cm，6.5mS/cm；pH5.5，电导4.5mS/cm、6.5mS/cm以及pH5.8，电导4.5mS/cm，收集各条件样品进行检测(见图5)。由于低于pH5.2的酸沉条件对hGH的细胞活性和目的蛋白回收有明显影响；综合pH5.0上样时洗脱液中目的蛋白纯度的明显降低，认为上样pH值应在pH5.5和pH5.8中选取；pH5.8，4.5mS/cm上样时，载量较低，约为8CV，但纯化后目的蛋白回收较高；pH5.5，4.5mS/cm上样时，载量大于20CV，纯化后的目的蛋白纯度稍有降低，可作为优选的上样条件。MMC层析作为第一步提纯，目的蛋白的回收更为重要，据此确定pH5.5，电导4.5mS/cm为优选的上样条件。
4.MMC层析洗脱条件的确定
采用pH5.8的上样条件，再平衡后，先用pH6.3的缓冲液提高MMC柱的pH值后，在50mMTris，50mM NaAC，pH6.3的缓冲液体系下进行升盐洗脱，0～2MNaCl，20CV，收集各样品进行检测。结果显示50mMTris，50mM NaAC，pH6.3可将大部分目的蛋白洗脱下来，洗脱液纯度改善明显(如图6)；在50mM Tris，50mM NaAC，pH 6.3的基础上，对pH和盐浓度进行改变，测试了：50mM Tris，50mM NaAC，pH 6.5；50mM Tris，50mM NaAC，100Mm NaCl，pH6.3；50mM Tris，50mMNaAC，pH6.3等三种条件进行洗脱，观察MMC层析的去杂效果。如图7结果所示，50mM Tris，50mM NaAC，pH6.3作为洗脱液效果最佳，可通过增加洗脱液的体积来提高收率。
5.MMC载量的确定
取80g OsrhGH米粉加入400ml提取液，搅拌提取1h后，酸沉2h。采用Tricorn10层析柱，柱体积5ml，上样流速1.5ml/min；上样约50mL时开始采用分部收集器按照10ml即2CV开始自动收集，对收集的样品进行SDS-PAGE及Western Blot检测。结果显示第24管有信号出现，即MMC层析柱的最大载量为24×2+10＝58CV，去除管道死体积，约55CV。若按照80％的载量算，最终确定MMC的上样量为40CV(图8)。
6.MMC层析条件的确定
采用4～6个柱体积(CV)的pH为5.4～5.6的50mM Tris、50mM NaAc的缓冲液，以170～240cm/h的线性流速平衡层析柱；以实施例1的调配液为层析上样液，其中上样液pH为5.4～5.6、电导3.5～4.5mS/cm，上样体积20～40CV； 用pH为6.2～6.4的50mM Tris、50mM NaAc缓冲液，以170～240cm/h的线性流速进行洗脱，收集含有OsrhGH的洗脱液，获得初级纯度的OsrhGH。
【实施例3】通过Q FF阴离子交换层析进行中级纯化
1.Q FF层析最大载量的测定
将MMC层析上样量为40CV的层析洗脱液倍比稀释后，调节pH至7.2～7.4，作为Q FF层析载量测定的上样液。采用1mL Q FF层析柱进行测定，流速0.25mL/min。收集穿透液，并进行SDS-PAGE及Western Blot检测(图9)，结果显示Q FF层析的最大载量在50CV(即泳道20)和70CV(即泳道30)之间。根据80％的最大载量上样，其上样量为40CV。
2.Q FF层析条件的确定
经过不断优化，最终确定Q FF层析的条件如下：采用10～15个柱体积的pH为7.2～7.4的25mM Tris-HCl缓冲液，以120～300cm/h的流速平衡柱子；将步骤2的含OsrhGH的洗脱液中加入约等体积的纯水，并调节pH至7.2～7.4，作为此步层析的上样液，其中样品电导为3～3.4mS/cm；用4～6个柱体积、pH为7.2～7.4、电导5.5～6.0mS/cm的25mM Tris-HCl，35mM NaCl缓冲液以120～300cm/h的流速进行杂蛋白的洗脱；用4～6个柱体积、pH为7.2～7.4、电导8.1～8.6mS/cm的25mM Tris-HCl，60mM NaCl缓冲液以120～300cm/h的流速进行OsrhGH的洗脱，获得中级纯度的OsrhGH。
【实施例4】通过疏水层析进行精纯
1.疏水层析介质的确定
将实施例3中Q FF层析洗脱样品，加入终浓度为1M的硫酸铵，调节pH至7.5即为上样液。将层析上样液分别上至柱体积均为5mL的phenyl HP，Octyl FF层析柱，上样流速1.25mL/min，上样体积为50mL；采用线性洗脱的方式进行洗脱。SDS-PAGE结果显示(图10)，Phenyl HP层析的8号样下面几乎无降解带(图10所示)，目的蛋白主要集中在洗脱峰主峰的前半部分，9号、10号降解带开始大量被洗脱。Octyl FF层析线性洗脱，由于分辨率不如phenyl HP，只有一个主峰，降解蛋白主要在6号样即主峰的前半部分存在。优化空间不大。因此初步确定Phenyl HP层析作为精纯步骤。
2.phenyl HP层析的优化
确定Phenyl HP介质后，分别对Phenyl HP层析条件进行如下优化：(1)1M硫酸铵上样。0.5M硫酸铵(含10mM PB，pH 7.5)洗杂，0～0.5M硫酸铵(含10mM PB，pH 7.5)线性洗脱；(2)0.5M硫酸铵上样，0.25M硫酸铵洗脱；(3)1M硫酸铵上样，0.5M硫酸铵，0.25M硫酸铵，0.125M硫酸铵(含10mM PB， pH 7.5)梯度洗脱。通过比较3个优化条件下的层析图谱及SDS-PAGE检测结果(图11、图12)，最终确定Phenyl HP层析选择0.5M硫酸铵浓度上样，洗脱液硫酸铵浓度约在0.25M左右。
3.Q FF层析不同上样量对Phenyl HP层析行为的影响
根据前期的试验结果，当Q层析上样量40CV时，其层析行为已经发生变化，后续的Phenyl HP的层析行为虽然没有变化，但是样品的纯度明显降低。为此，对Q FF层析及Phenyl HP层析的上样体积进行如下设计：(1)将Q FF上样10CV层析洗脱液按照10CV上样Phenyl HP层析；(2)将Q FF上样20CV层析洗脱液按照5CV上样Phenyl HP；(3)将Q FF上样20CV层析的洗脱液按照10CV上样Phenyl HP；结果显示Q FF 20CV、Phenyl HP 5CV与Q FF 10CV Phenyl HP 10CV的洗脱样品纯度较高，明显比Q FF 20CV和Phenyl HP 10CV的高(见图13)。综合成本等因素，最终选择Q FF的上样量为20CV，Phenyl HP的上样量为5CV。
4.Phenyl HP层析条件的确定
用4～6倍柱体积的pH为7.2～7.4的电导为73～76mS/cm的8～12mM磷酸钠、0.5M硫酸铵的缓冲液以50～180cm/h的流速平衡柱子。将Q FF中级纯度的洗脱样品采用3mM硫酸铵调节电导至75～78mS/cm，pH调节至7.2～7.4，作为此步层析的上样液；用pH为7.2～7.4的8～12mM磷酸钠、0.5M硫酸铵缓冲液以50～180cm/h的流速洗脱样品，收集含有重组人生长激素的洗脱液，获得纯度95％以上的的重组人生长激素。
【实施例5】小试工艺验证
根据实施例1～4确定的工艺条件，对小试工艺进行了多批验证，确定了工艺的可行性(如图14～16)，经过3步纯化后产量可达87mg/kg糙米(表2)。同时产品的内毒素含量＜5EU/mg(表3)、经SEC-HPLC测试单体纯度＞97％(表4，图17)，宿主DNA残留＜1ng/mg(表5)，均符合2015版中国药典标准。在宿主蛋白残留方面，目前暂时高于药典标准，但是由于表达体系不同，宿主蛋白的毒性也不同，后期将进一步研究宿主蛋白去除及可能的毒性作用。另外在生物活性方面，OsrhGH与中检院标准品的活性相当，甚至更高(图18)。
表2 三批层析收率
Figure PCTCN2019110385-appb-000002
表3 OsrhGH内毒素检测结果
项目 201705001 201705002 201705003
内毒素 2.2～4.4EU/mg 1.1～2.2EU/mg 1～2EU/mg
表4 4批OsrhGH的SEC～HPLC检测结果
项目 标准品 001批 002批 003批 004批
hGH单体 99.250 98.061 98.374 97.779 98.223
hGH二聚体 0.554 1.246 1.124 1.470 1.185
hGH 99.804 99.307 99.498 99.249 99.408
表5 OsrhGH中OHCP、DNA残留检测
项目 201706001 201706002 201706003 201706004
HCP残留 8.3ug/mg 12.7μg/mg 4.5μg/mg 5.7μg/ml
DNA残留 0.22ng/mg 0.55ng/mg 0.63ng/mg 0.38ng/mg

Claims (11)

  1. 一种从基因工程水稻种子中分离纯化重组人生长激素的方法，依次包含以下步骤：
    1)从重组人生长激素基因工程水稻种子中提取含有重组人生长激素的粗提取物；
    2)将含有重组人生长激素的粗提取物经Bestarose Diamond MMC阳离子交换层析分离纯化，得到初级产物I；
    3)将初级产物I经Q Sepharose FF阴离子交换层析分离纯化，得到含有重组人生长激素的中级产物II；
    4)将中级产物II经phenyl HP疏水层析分离纯化，得到纯化的重组人生长激素目标物。
  2. 根据权利要求1所述的方法，其特征在于，步骤1)所述重组人生长激素粗提取物是通过下述方法制备：
    i)将含有重组人生长激素的基因工程水稻种子粉碎后与提取缓冲液混合，在4～30℃下提取1～2小时；所述提取缓冲液包含5～25mM Tris，5～25mM NaAC，pH为5.5～7.5；
    ii)将步骤i)的提取混合物的pH调节至5.0～5.8，并沉淀1～4小时；
    iii)过滤步骤ii)的混合物，收集滤液，获得含有高浓度重组人生长激素的粗提物溶液。
  3. 根据权利要求1所述的方法，其特征在于，所述步骤2)中，Bestarose Diamond MMC阳离子交换层析采用4～6倍柱体积的pH为5.4～5.6的50mM Tris、50mM NaAc的缓冲液，以170～240cm/h的线性流速平衡层析柱；以步骤1)获得的粗提取物为层析上样液，上样缓冲液的pH为5.2～5.8，电导为3.5～5mS/cm；所述Bestarose Diamond MMC阳离子层析中洗脱目标物的缓冲液包含50mM Tris、50mM NaAC，pH为6.2～6.4。
  4. 根据权利要求3所述的方法，其特征在于，所述Bestarose Diamond MMC阳离子交换层析的上样缓冲液的pH为5.2～5.8，电导为4～5mS/cm，上样量为20～40倍柱体积。
  5. 根据权利要求1所述的方法，其特征在于，所述步骤3)中，Q Sepharose FF阴离子层析采用10～15倍柱体积的pH为7.0～7.6的25mM Tris-HCl缓冲液，以120～300cm/h的流速平衡柱子；所述Q Sepharose FF层析中的上样液为步骤2)中MMC阳离子层析洗脱得到的初级产物I调配而成，调配方法为在所述初级产物I中加入等体积的纯水，并调节pH至7.0～7.6，电导为3～3.4mS/cm。
  6. 根据权利要求1所述的方法，其特征在于，所述Q Sepharose FF阴离子层析中，洗脱杂蛋白的缓冲液包含5～25mM Tris-HCl缓冲液或磷酸盐缓冲液，30～40mM NaCl溶液，pH为7.0～7.5；所述Q Sepharose FF阴离子层析洗脱重组人生长激素的缓冲液包含5～25mM Tris-HCl，60mM NaCl溶液，pH为7.0～7.5。
  7. 根据权利要求6所述的方法，其特征在于，所述Q Sepharose FF阴离子 层析使用的洗脱缓冲液包含：洗脱杂蛋白的缓冲液为25mM Tris-HCl，35mM NaCl缓冲液，pH为7.2～7.4，电导为5.5～6.0mS/cm；以及，
    洗脱重组人生长激素的缓冲液为25mM Tris-HCl，60mM NaCl缓冲液，pH为7.2～7.4，电导为8.1～8.6mS/cm。
  8. 根据权利要求1所述的方法，其特征在于，所述步骤4)中，phenyl Bestarose HP层析采用4～6倍柱体积的pH为7.2～7.4，电导为73～76mS/cm的8～12mM磷酸钠、0.5M硫酸铵的缓冲液以50～180cm/h的流速平衡柱子；上样液为Q Sepharose FF层析洗脱液即中级产物II调配而成，调配方法为在所述Q Sepharose FF层析洗脱液中加入终浓度0.5～1M的硫酸铵，并调节pH至7.2～7.4。
  9. 根据权利要求8所述的方法，其特征在于，所述Q Sepharose FF层析的上样量为20倍柱体积，Phenyl Bestarose HP层析的上样量为5倍柱体积。
  10. 根据权利要求8所述的方法，其特征在于所述Phenyl Bestarose HP层析洗脱缓冲液为含有8～12mM磷酸钠，0.5M硫酸铵的缓冲液，pH为7.2～7.4。
  11. 根据权利要求1所述的方法，包括下述步骤：
    a)制备OsrhGH的粗提取液：
    将重组人生长激素基因工程水稻种子脱壳加工成半精米，研磨成80～100目的米粉，将米粉与提取缓冲液以重量/体积，kg/L为1:5的比例混合，于25～28℃提取获得混合物；提取缓冲液的成分为：25mM Tris-乙酸，25mMNaAc，pH7.5；
    将上述得到的混合物用20％乙酸调节pH至5.4～5.6，放置沉淀后，加入2～5％的珍珠岩进行压滤，压滤完毕后复调pH至5.4～5.6，0.22μm滤膜过滤后即为OsrhGH的粗提取液；
    b)将所述OsrhGH的粗提取液通过阳离子交换层析进行初级纯化：
    层析介质为Bestarose Diamond MMC，采用4～6倍柱体积(CV)的pH为5.2～5.6的50mM Tris、50mM NaAc的缓冲液，以170～240cm/h的线性流速平衡层析柱；以步骤a)的粗提物为层析上样液，其中上样液pH为5.2～5.6、电导为4～5mS/cm；上样体积20～40CV；用pH为6.0～6.5的50mM Tris、50mM NaAc缓冲液，以170～240cm/h的线性流速进行洗脱，收集含有OsrhGH的洗脱液，获得含OsrhGH的初级产物I；
    c)将所述初级产物I通过阴离子交换层析进行中级纯化：
    层析介质为Q Sepharose FF，采用10～15倍柱体积的pH为7.0～7.5的25mM Tris-HCl缓冲液，以120～300cm/h的流速平衡柱子；将步骤b)的含OsrhGH的洗脱液中加入约等体积的纯水，并调节pH至7.2～7.4，作为此步层析的上样液，其中样品电导为3～3.4mS/cm；用4～6倍柱体积，pH为7.2～7.4，电导5.5～6.0mS/cm的25mM Tris-HCl，35mM NaCl缓冲液以120～300cm/h的流速进行杂蛋白的洗脱；用4～6倍柱体积，pH为7.2～7.4，电导8.1～8.6mS/cm的25mM Tris-HCl，60mM NaCl缓冲液以120～300cm/h的流速进行OsrhGH的洗脱，获得含OsrhGH的中级产物II；
    d)采用疏水层析进行精纯：
    层析介质为Phenyl Bestarose HP，用4～6倍柱体积的pH为7.2～7.4，电导为73～76mS/cm的8～12mM磷酸钠、0.5M硫酸铵的缓冲液以50～180cm/h的流速平衡柱子；将步骤c)得到的中级产物II采用3mM硫酸铵调节电导至75～78mS/cm，pH调节至7.2～7.4，作为此步层析的上样液；用pH为7.2～7.4的8～12mM磷酸钠、0.5M硫酸铵缓冲液以50～180cm/h的流速洗脱样品，收集含有重组人生长激素的洗脱液，获得纯度95％以上的的重组人生长激素。
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